Fluorescence Resonance Energy Transfer (FRET) is a process whereby a first fluorescent dye (the “donor” dye) is excited, typically by illumination, and transfers its absorbed energy to a second dye (the “acceptor” dye) that has a longer wavelength and therefore lower energy emission. Where the second dye is fluorescent, energy transfer results in fluorescence emission at the wavelength of the second dye. However, where the second dye is non-fluorescent, the absorbed energy does not result in fluorescence emission, and the fluorescence of the initial donor dye is said to be “quenched”. The proximity of the donor and acceptor molecule and the overlap in spectra of the energy emitted by the donor and the absorption spectra of the acceptor molecule are critical for efficient energy transfer.
When the acceptor molecule is a quencher, the FRET pair can be used in assays to detect the interaction, assembly, cleavage, dissociation and conformation of proteins, nucleic acids and other biomolecules. This can be accomplished when the donor and acceptor are conjugated to different substances wherein the resulting signal is determined by the proximity of the donor and acceptor molecules. Cleavable conjugates are also widely used to detect proteases wherein the donor and acceptor are conjugated to the same substance and the appropriate protease cleaves a bond that changes the proximity of the donor and acceptor (U.S. Pat. Nos. 6,323,337; 6,348,596 and 6,399,392).
In addition, FRET is used in bioassays wherein it is beneficial to have energy emitted at a longer wavelength that does not interfere with the autofluorescence of the bioassay sample. Typically in these bioassays a FRET pair, commonly referred to as a tandem, is conjugated to an antibody or other ligand for the detection of an analyte. These tandem conjugates are also widely used with flow cytometers and confocal laser scanning microscopes that are equipped with separate epifluorescence filter sets wherein a larger Stokes shift is beneficial for multicolor applications. Phycobiliproteins are well known fluorescent proteins that are used as a donor conjugated to a protein such as an antibody wherein a xanthene based dye such as Texas Red (Molecular Probes, Inc.) or a cyanine dye is used as the acceptor (U.S. Pat. Nos. 4,859,582 and 4,542,104; RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS; 9thedition, CD-ROM, 2002). In this way, with the Texas Red conjugate of R-phycoerythrin (R-PE), the resulting tandem conjugate can be excited at 488 nm using the widely available argon-ion laser and the energy is re-emitted at about 620 nm or longer after having been transferred to the acceptor dye. Importantly, this permits the simultaneous use of a second conjugate of R-PE that is not a tandem conjugate as a label for a different target. These tandem conjugates find wide use in bioassays; however, in systems that utilize flow cytometry the signal from the fluorescent protein is not fully transferred to the acceptor and therefore must be compensated for when determining multiple targets with labels that absorb and emit in the same range as the fluorescent protein. Thus, there is a need to reduce this compensation determining multiple targets in a sample using R-PE tandem conjugates.
The idea of using a quencher to form a ternary conjugate wherein the quencher compound accepts the residual energy of the R-PE donor compound goes against the current teaching and understanding of FRET. However, we have unexpectedly demonstrated that a compound absorbing in the emitting range of the R-PE can accept the residual fluorescense of the fluorescent protein. This unexpected improvement allows for detection of multiple targets using R-PE tandem conjugates by lowering the need for compensation. Ideally these compounds will be non-fluorescent, or essentially non-fluorescent, and will accept energy in the range that the donor emits the residual fluorescence.
The compounds of the instant invention represent a new and highly useful class of non-fluorescent energy acceptors, including chemically reactive versions, and the conjugates prepared therefrom.